Mobile phase preparation device for liquid chromatography

ABSTRACT

A mobile phase delivery device for use with high pressure liquid chromatography includes a manifold having a plurality of inlets, wherein each inlet is fluidly coupled with a solvent source. The manifold further has a mobile phase outlet that outputs a mobile phase, composed of solvent or solvent mixture, onto an analytical line. The device further includes at least one pump for delivering solvent to the manifold, and a fluid branch in the manifold that is located at a point downstream of the plurality of inlets, and is fluidly coupled with at least one of a pressure sensor and a purge valve.

BACKGROUND

The invention relates generally to a mobile phase preparation device foruse with liquid chromatography, in particular high pressure liquidchromatography (HPLC) or ultra-high pressure liquid chromatography(UHPLC). Basically, liquid chromatography (LC) is a physical method ofseparation in which the components to be separated are distributedbetween a mobile phase and a stationary phase, through or along whichthe mobile phase percolates in a definite direction.

The field of liquid chromatography started with the use of gravitypressure to move liquids through columns packed with a large particlesolid phase support, such as with 20 to 50 micrometers average particlesize, in order to separate molecules. In the 1970s, high pressure liquidchromatography was introduced to provide better resolution and fasteranalyses by using pumps to move liquids through columns packed withsmall particles (three to ten micrometers) at pressures up to 6,000 psi(˜4.1×10⁷ Pascal). Over the past decade, ultra-high pressure liquidchromatography, pioneered by James W. Jorgenson and co-workers in thelate 1990s (see, for instance, Anal. Chem. 1997, 69, 983-989), hasbecome very popular, as it provides even higher resolution andthroughput, but requires pumps that move liquids through columns packedwith very small particles (less than two micrometers) at pressures up to15,000 psi (˜1.0×10⁸ Pascal) and more. In order to achieve the maximumbenefits of UHPLC, instrumentation needed to be redesigned to withstandthese higher pressures, but also to minimize system volumes whichadversely impact resolution, throughput and reproducibility at UHPLCpressures.

Modern commercially available HPLC and UHPLC systems have a pressuretransducer and a purge valve in the fluid path of each solventdelivering pump prior to combining the solvents in a manifold (see fluidschematic in FIG. 1). These components, especially the pressuretransducer, can add a significant volume to the fluid path between thepump outlet and the manifold inlet, generally between 500 to 1,000microliters. Although systems can use software to compensate for thecompressibility of the different solvents used in each pump, for exampleby speeding up the pump to compress the solvent, this softwarecompensation gets more difficult to do accurately as the volume ofsolvent to be compressed increases. At extremes of a gradientseparation, characterized by continuous or stepwise change of thecomposition of the mobile phase during the elution process, such as 0 to5 percent of one solvent and, correspondingly, 100 to 95 percent ofanother solvent in a binary system, the solvent from the higher flowingpump can actually flow into the compression region of the lower flowingpump to compress its solvent to the system pressure. This cross flow ofsolvent can result in inaccurate solvent gradients and/or result inlonger re-equilibration times between runs to insure the proper solventmixture is being proportioned into the mixer at the start of a gradientrun.

Although many UHPLC system components have been designed withpre-combination solvent compression in mind, there has not been anyprevious development that combined multiple system components toproperly address these issues.

SUMMARY

In accordance with the principles of the invention, a first embodimentcomprises a manifold having a plurality of inlets, wherein each inlet isconfigured to be fluidly coupled with a solvent source, and furtherhaving a mobile phase outlet configured to output a mobile phase,composed of solvent or solvent mixture, onto an analytical line, atleast one pump for delivering solvent to the manifold, and a branchlocated at a point downstream of the plurality of inlets that is fluidlycoupled with at least one of a pressure sensor and a purge valve.

Integrating at least one of a pressure sensor and purge valve within asolvent mixing device improves the overall performance of a UHPLCsystem. By locating at least one of these components at, or downstreamof, a point where the solvents combine to form the mobile phase, insteadof placing the component before the manifold as known from the priorart, the post pump compression volume can be reduced by a factor of 10to 100, which greatly reduces system volume and minimizes solventcompressibility issues. Under pressure, solvents undergo compression andthis solvent compression, which is different for all solvents andincreases as pressure increases, can have a significant impact on theflow rate of the solvents being delivered by UHPLC pumps. By minimizingthe volume of solvent to be compressed, the software procedures appliedto correct for compressibility, a task generally challenging toimplement dynamically as system pressures change during gradient elutionUHPLC, can be downscaled, if not dispensed with completely. With systemsconstructed in accordance with principles of the current invention, thecompression volume can be considerably reduced, such as to less than tenmicroliters, which greatly reduces the impact of solvent compression onthe actual flow of the pumps versus the programmed flow.

The analytical line or analytical conduit is generally characterized inthat it transmits the mobile phase to be used in a chromatographic runand, to that end, directly or indirectly leads to a chromatographicseparation column. On its way there, it passes a point at which liquidsample is introduced into the mobile phase, preferably in a pulse withnarrow spatial distribution as to allow for high chromatographicresolution.

The mobile phase may comprise a mixture of a plurality of solvents andis formed generally by combining the different solvents, even if, as aresult of the confluence, the distribution of the solvents in the liquidvolume of the mixture is not initially homogeneous. Under certaincircumstances, however, the mobile phase may also comprise just onesolvent, for instance in cases where a gradient elution schedule at somepoint includes reducing a percentage of one solvent to zero whereasraising the percentage of the other complementary solvent to 100.

In various embodiments, the mobile phase preparation device furthercomprises a distribution homogenizer fluidly coupled on the analyticalline to the mobile phase outlet and being configured to homogenize, asthe case may be, an inhomogeneous distribution of solvents in the mobilephase. The distribution homogenizer may comprise a mixer cartridge, forexample. Mobile phase preparation devices constructed according toprinciples of the invention allow the pressure sensor, optionally purgevalve and/or distribution homogenizer to be built in close spatialproximity into a single block, such as a solvent mixing device, withcustomized machining to reduce volumes of each device and eliminatevolumes between devices, as well as to reduce the number of ultra-highpressure fluidic connections.

In various embodiments, the at least one pump is a single-piston pump.Preferably, the single-piston pump has a displacement volume of about 15microliters. One pump can be employed to deliver a plurality of solventsfrom the solvent sources to the manifold when the total aspiration powergenerated by the pump is distributed over several different solventdelivery lines. In other embodiments, however, it is also possible touse a plurality of pumps, for example such that each solvent isdelivered by an associated pump.

In various embodiments, the pressure sensor is configured to operate upto about 20,000 psi (corresponding to about 1.4×10⁸ Pascal) tofacilitate measurements under ultra-high pressure liquid chromatographyconditions. Preferably, the purge valve, if present, is fluidly coupledto the pressure sensor downstream thereof.

In various embodiments, the pressure sensor is a pressure transducer.The pressure transducer may comprise a diaphragm sensor that measuresfluid displacement. The pressure sensor is fluidly coupled to the flowof mobile phase in a branch off the analytical line and monitors theoverall pressure in the mixing device preferably somewhere between apoint where the different solvents combine to form the mobile phase, upto a sample injector. If a distribution homogenizer is present, it ispreferred to plumb the branch into the analytical line somewhere betweenthe point of confluence and the inlet of the distribution homogenizer asto facilitate a compact and high pressure resistant instrumentalconfiguration.

In various embodiments, the branch includes an outlet at the manifoldoff the analytical line. In some embodiments, the manifold has twoinlets for receiving two solvents to implement a binary gradient elutionsystem, the two inlets and the two outlets being coupled in a crossconnection. The cross connection can be a cross fitting. However, thisdescription is not to be construed restrictive. The four arms of thecross connection, or of any other multi-legged manifold, do not have tolie in the same plane. Rather, it is possible to conceive designs ofconnecting four lines that distinguish from a standard cross fitting.

In various embodiments, the mobile phase preparation device furthercomprises a controller that operates the at least one pump such thatpumping powers for different solvents change over time as to vary acomposition of the mobile phase. With such configuration it becomespossible to operate the mobile phase preparation device with gradientelution in order to achieve high flexibility for different analyticalinvestigations.

In various embodiments, the mobile phase preparation device furthercomprises tubing that fluidly connects an outlet of a pump directly toan inlet of the manifold, wherein a length of the tubing is minimized asto reduce fluid volume between pump and manifold. Consequently, nopressure sensors and/or purge valves, which would add up to thecompression volume associated with the respective pump, are located inthe solvent delivery line between pump outlet and manifold inlet. In sodoing, system performance can be considerably improved.

In various embodiments, the mobile phase preparation device furthercomprises a controller that acquires pressure measurement data from thepressure sensor and adjusts operation of the at least one pump as afunction of the pressure measurement data.

The main achievement of this invention is the reduction of total systemvolume which improves throughput and overall performance of a UHPLCsystem. By reducing the volume of the solvents to be compressed at UHPLCpressures prior to combining them to form the mobile phase, the systemis able to achieve the desired flows from the pumps much more rapidly,thus resulting in faster equilibration between runs. This lower volumeof solvent compression also minimizes any cross contamination betweensolvents with different compressibility, which improves both thethroughput and reproducibility of the UHPLC system for gradientanalysis.

In some embodiments, the mobile phase preparation device furthercomprises a mechanism by which a number of high pressure resistantfluidic connections on fluid lines is minimized. A preferred way ofrealizing such mechanism is incorporating the manifold, the pressuresensor, optionally the purge valve, as the case may be the distributionhomogenizer, a portion of the analytical line, and generally theassociated fluid lines in close spatial proximity into a single block ofpressure resistant material. Thus, the total number of connectionsrequired to integrate pressure sensors and/or purge valves into a UHPLCsystem can be reduced, which minimizes the opportunity for solvent leaksat high pressures, improving the overall reliability of the UHPLCsystem. Visual inspection of these high pressure connections and directaccess to them for tightening as required is also possible, with noopportunity for solvent leaks to go undetected or to allow solvents toleak into the electronics of the UHPLC system.

In a second aspect, the invention pertains to a method of operating amobile phase preparation device during a run of high pressure liquidchromatography, comprising the steps: (i) pumping a solvent from asolvent source to an inlet of a manifold; (ii) passing the solventthrough a confluence region within the manifold where the solvent can becombined with at least one other solvent, being delivered to themanifold through a different line and inlet, to form a mobile phase;(iii) outputting the resultant mobile phase from the manifold onto ananalytical line; and (iv) branching off a portion from the flow ofmobile phase at, or downstream from, the confluence region in order toperform at least one of a pressure measurement and venting to exhaust.

In various embodiments, a result of the pressure measurement is used tocontrol the pumping.

In various embodiments, in a binary, tertiary, or quaternary mode ofoperation, a first solvent is combined with one, two, and three othersolvents, respectively, to form the mobile phase.

In a third aspect the invention relates to a solvent mixing device foruse with high pressure liquid chromatography, comprising a manifoldhaving a plurality of inlets, wherein each inlet is configured to befluidly coupled with a solvent source, and further having a mobile phaseoutlet configured to output a mobile phase, composed of solvent orsolvent mixture, onto an analytical conduit, and a dead-ended conduitbeing plumbed in at a point downstream from the plurality of inlets, andbeing fluidly coupled with at least one of a pressure sensor and a purgevalve.

In various embodiments, the solvent mixing device further comprises ablock of high pressure resistant material, such as stainless steel ortitanium, into which at least the manifold, the dead-ended conduit, aportion of the analytical conduit, at least one of the pressure sensorand purge valve, and corresponding fluid lines are incorporated, suchthat high pressure resistant fluidic connections at coupling points offluid lines are dispensable. The only high pressure resistant fluidicconnections that remain are those where the block is fluidly coupled tothe outside, such as at the manifold inlets and at the analytical lineoutlet, or as the case may be at a homogenizer outlet.

In some embodiments, the solvent mixing device further comprises adistribution homogenizer with its fluid lines being incorporated intothe block, and being plumbed in to the portion of the analyticalconduit.

Preferably, the solvent mixing device comprising a single block of highpressure resistant material has the configuration of a module and can beeasily disassembled from, and reinserted into, a mobile phasepreparation device, for example, for maintenance and/or cleaningpurposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood by referring to the followingfigures. The elements in the figures are not necessarily to scale,emphasis instead being placed upon illustrating the principles of theinvention (often schematically). In the figures, like reference numeralsgenerally designate corresponding parts throughout the different views.

FIG. 1 illustrates schematically a conventional mobile phase preparationdevice;

FIG. 2 illustrates schematically a mobile phase preparation deviceaccording to principles of the present invention;

FIGS. 3A-B illustrate schematically mobile phase preparation devicesaccording to principles of the present invention with tertiary andquaternary configurations, respectively;

FIG. 4 illustrates another embodiment of a binary mobile phasepreparation device according to principles of the invention; and

FIG. 5 shows schematically a liquid chromatography set-up which mayincorporate a mobile phase preparation device according to principles ofthe invention.

FIG. 6 shows the steps in an illustrative method for operating a liquidchromatography system in accordance with the principles of theinvention.

DETAILED DESCRIPTION

While the invention has been shown and described with reference to anumber of embodiments thereof, it will be recognized by those skilled inthe art that various changes in form and detail may be made hereinwithout departing from the spirit and scope of the invention as definedby the appended claims.

FIG. 1 shows a fluid schematic of a binary mobile phase preparationdevice, that is, with input of two solvents A and B, constructed inaccordance with the prior art. Each reservoir is fluidly coupled to apump 2A,B each of which is configured and controlled to deliver solventfrom the corresponding reservoir on a solvent delivery line 4A,B to amixing device 6. The components the solvent further passes online willnow be explained in the following in more detail.

Pressure transducers 8A,B monitor the pressure generated at each pumpoutlet. In addition to monitoring the operation of the pumps 2A,B, thepressure output of the transducer 8A,B usually is tied into the pumpflow to provide feedback to the pump 2A,B and insure pressures are inthe correct range. This component typically uses a diaphragm sensor thatmeasures fluid displacement, and therefore the volume of this deviceusually changes with changing pressures. Some HPLC and UHPLC systemsutilize a pulse dampener (not shown) in addition to the pressuretransducer. In other implementations, the pressure transducer 8A,Bitself can also serve as a pulse dampener, minimizing short termpressure fluctuations due to the large volume and compression of thediaphragm. The pressure transducer 8A,B generally is the largest volumeof any of the components between pump outlet and manifold inlet. Asthere is a separate pressure transducer 8A,B for each pump 2A,B, each ofthese adds its inherent volume to the fluid path of each pump 2A,B sothat a large volume results and this volume must be compressed beforethe correct flow for each pump 2A,B can be realized.

Each of the pressure transducers 8A,B is fluidly coupled to a purgevalve 10A,B downstream. These purge valves 10A,B allow the pumps 2A,B tobe primed and to rapidly change solvents in each pump 2A,B as required.In lab practice, this is usually done once a day. The volume of thepurge valve 10A,B typically is not as large as the volume of thepressure transducer 8A,B, but still perceptibly contributes to theoverall fluid volume between pump outlet and manifold inlet.Furthermore, the valve 10A,B requires two additional high pressureconnections between pump outlet and manifold. These high pressureconnections are potentially weak spots in a highly pressurized system,the failure of which could pose a threat to surrounding instrumentationand lab personnel.

The Tee fitting 6 combines the flows from the two pumps 2A,B prior to amixer cartridge 12. During operation, the mixer cartridge 12 homogenizesefficiently, as the case may be, any inhomogeneously distributedsolvents in the mobile phase to insure a smooth baseline and retentiontime reproducibility for different chromatographic runs. Severaldifferent mixer designs are available on the market, depending mainly onthe pumps and associated volumes, and are therefore known to one ofordinary skill in the art.

From the mixer cartridge 12 the homogenized mobile phase, after passingan injector module (not shown) for injecting sample into the mobilephase stream, is usually delivered to the inlet of a column (not shown)where chromatographic separation takes place, and from there on to adetector (not shown).

FIG. 2 shows a first embodiment of a binary mobile phase preparationdevice according to principles of the present invention in a fluidschematic view. As can be seen, the outlets of the pumps 202A,B aredirectly fluidly connected to the respective inlet of cross connection206 without any intermediate components, such as pressure transducersand/or purge valves, which would increase the system volume betweenpumps 202A,B and cross 206 and give rise to afore-discussedcompressibility issues. Instead, the present embodiment comprises onlyone pressure transducer 208 plumbed in offline (in the sense of ‘off theanalytical line’) to the flow from each pump 202A,B to mixer cartridge212, so its volume does not add up to the compression volume of eachpump 202A,B. Preferably, the pressure readings of the pressuretransducer 208 are used by a controller to adjust operation of the pumps202A,B. Due to the reduced compression volume between each pump 202A,Band cross 206 the software correction for different solventcompressibility is less important and can be implemented in a lesscomplex manner.

Moreover, the present embodiment has a single purge valve 210, againplumbed in offline to the flow from the pump outlets to the mixercartridge 212. One outlet of the single purge valve 210 is closed asindicated by the cross. In principle, it provides the same function asindividual purge valves, such as purging and venting to exhaust, butwithout the extra volume and additional high pressure fittings on thesolvent delivery line between pumps 202A,B and cross 206.

In the embodiment shown, cross connection 206 replaces the Tee fittingshown in the prior art schematic of FIG. 1. The additional fourth leg ofthe cross connection 206 is a dead ended line through the pressuretransducer 208 to the purge valve 210. This arrangement allows thepressure transducer 208 and purge valve 210 to operate properly withinthe system, but keeps them out of the fluid streams between the pumpoutlets and the cross 206. Using this arrangement, it is possible toreduce the volume of fluid to be compressed under high pressures to lessthan ten microliters. In comparison, in conventional HPLC and UHPLCsystems as shown schematically in FIG. 1 this fluid compression volumefor each pump can amount to 500 to 1,000 microliters or more.

The mixer cartridge 212 arranged on the analytical line between themobile phase outlet of the cross connection 206 and a chromatographiccolumn (not shown) preferably is also chosen to have low volume. Itshould be well suited for flow ranges typically used in analytical LCsystems, such as ranging from 100 to 5,000 microliters per minute, andshould have good sweep out characteristics. With such characteristics,good performance and throughput of HPLC and UHPLC systems can beachieved.

For the sake of completeness, in the present embodiment, Luer fitting214 is generally attached to the waste leg of the purge valve 210 (on alow pressure side) to allow the user to attach a syringe to rapidly pullfresh solvents through the pumps 202A,B for priming and solventchangeover. Due to its arrangement offline, however, it does notcontribute to the decisive compression volumes of the pumps 202A,B.

Generally, the invention removes large solvent volume components fromthe solvent stream between the pump outlets and the manifold, forexample by plumbing them in to a single manifold at one leg thereof sothat only a single pressure transducer and purge valve are plumbed inoffline to the mobile phase flows as depicted in FIG. 2. Although thesecomponents, now located downstream compared to their prior artcounterparts, also have significant volumes, they are compressed by thetotal solvent flow from the pumps and there is no cross flow between thetwo or several solvent streams prior to the mixer cartridge. Thisresults in very accurate solvent compositions and rapid systemre-equilibration, allowing high throughput and reproducible gradientseparations independent of the relative solvent flows and systempressures.

In a particularly favorable embodiment, the cross connection 206, thepressure transducer 208, optionally the purge valve 210, and the mixercartridge 212 together with fluid lines are incorporated into a block ofhigh pressure resistant material (indicated by the dashed contour 240)in close spatial proximity to form a solvent mixing device, such thathigh pressure resistant fluidic connections at the fluid couplings ofthese components can be dispensed with. The block may be made fromstainless steel or titanium, for example.

Compared to prior art designs as represented by the fluid schematic ofFIG. 1, the mobile phase preparation device requires a lower totalnumber of high pressure resistant fluidic connections (represented bybold black dots). In the embodiment of FIG. 2 it is seven connections(two at pump inlets, two at pump outlets, two at manifold inlets, andone at mixer cartridge outlet) whereas the prior art design shown inFIG. 1 requires seventeen (two at pump inlets, two at pump outlets, fourat transducers, four at purge valves, three at Tee connection and two atmixer cartridge). In so doing, a risk of potential leaks that couldadversely impact system performance and pose danger for the surroundinginstrumentation as well as lab personnel is reduced. It goes withoutsaying that the number of fluidic connections would also be reduced evenif the afore-mentioned components, constituting an exemplary solventmixing device, were not incorporated into a single block. Then, however,the reduction would be less pronounced from seventeen to fourteen (whencomparing FIGS. 1 and 2).

The reduced solvent compression referred to above especially improvesgradient elution systems using two or more pumps to deliver two or moredifferent solvents. Albeit the principles of the present invention canalso be employed with mobile phase separation devices that use only onepump to deliver a plurality of solvents. In gradient elution, largesolvent compression volumes can result in cross flow of one solvent intothe post pump volume of the other solvent, resulting in significantchanges to the solvent composition versus what was programmed. This isespecially true at extremes of the solvent gradient, where one pump isrunning at significantly higher flow rates than the other pump(s). Thisproblem that usually manifests itself in impaired reproducibility of theretention time of sample compounds and/or in longer re-equilibrationtimes required to insure good performance can be overcome by methods anddevices configured in accordance with principles of the presentinvention.

FIGS. 3A-B show embodiments of the present invention where more than twosolvents are delivered to a solvent mixing device in order to constitutea mobile phase for liquid chromatography. With three solvents A,B,Cinvolved (FIG. 3A) the system is tertiary. With four solvents A,B,C,Dinvolved (FIG. 3B) the system is quaternary. As regards the othercomponents of the mobile phase preparation device the sameconfigurations can be employed. It is understood that the manifold hasto be slightly adapted to be able to receive more than two streams ofsolvent, for example, resembling a hub with several spikes. It goesfurther without saying that the number of solvents to be combined is inprinciple not limited to four. Systems exceeding that number are alsoconceivable and can be readily implemented by one of ordinary skill inthe art when being aware of the present disclosure.

FIG. 4 shows another fluid schematic of an embodiment of a binary mobilephase preparation device according to principles of the invention. Incontrast to the embodiment shown in FIG. 2, the present implementationcomprises two Tee connections 406A,B. The first Tee connection 406A hastwo inlets for receiving flows of the two solvents A,B delivered bypumps 402A,B, and one mobile phase outlet for outputting the mobilephase, resulting from the confluence of the two solvents A,B, onto theanalytical line. Instead of providing a branch in the confluence regionof the solvents A,B, it is implemented further downstream in the secondTee connection 406B. The second Tee connection 406B has one inlet forreceiving the mobile phase output from the first Tee connection 406A,one outlet for transmitting the mobile phase to the subsequent mixercartridge 412, and another outlet off the analytical line that isfluidly coupled to the inlet of single pressure transducer 408 and leadsonto a line which is in principle dead-ended. An advantage of thisexemplary implementation is that retrofitting a set-up as shown in theprior art schematic of FIG. 1 to comply with the principles of thepresent invention is easier, since the Tee connection 6 for combiningthe solvents A,B can remain in the device while the pressure transducers8A,B and purge valves 10A,B have to be removed and another Teeconnection 406B has to be plumbed in between the first Tee 406A and themixer cartridge 412.

If the two Tee connections 406A,B, the pressure transducer, optionallythe purge valve, and the mixer cartridge together with the respectivefluid lines are incorporated into a single block 440 so that highpressure resistant fluidic connections within the block are dispensable,the total number of high pressure resistant fluidic connections resultsin seven, actually the same number found with the implementation shownin FIG. 2. If no block design is foreseen, the total number will stillbe reduced, however, just by the smallest number of one fluidicconnection compared to the configuration shown in FIG. 1.

FIG. 5 shows a schematic of a high pressure liquid chromatographyseparation device where a mobile phase preparation device, and/or asolvent mixing device, according to principles of the invention can beemployed.

Solvents A,B (C,D) are provided in appropriate reservoirs, such as glasscontainers which can be tapped through the cap via flexible tubes toallow for quick and easy replacement thereof. If two solvents A,B arecombined to form a mobile phase, a binary gradient elution system can beoperated. If more solvents C,D (indicated by the dashed contours) areneeded, tertiary and quaternary systems can be implemented,respectively. A degasser (not shown) may be plumbed in to the solventdelivery lines in order to remove possible bubbles from the solventstreams, which, if present, could impair proper formation of a mobilephase with well-defined solvent ratios.

A mobile phase preparation device 520 is connected to the plurality ofsolvent sources and is responsible for delivering solvents A,B (C,D) toa mixer, combining them, homogenizing, as the case may be, aninhomogeneous distribution of solvents A,B (C,D) in the freshly formedmobile phase, and outputting the resultant homogeneous mobile phase ontoan analytical line. Downstream of the mobile phase preparation device520, high pressure is imposed on the mobile phase in order to drive itthrough the densely packed analytical column arranged furtherdownstream. The mobile phase preparation device 520 can be implementedin accordance with the principles of the present invention, and asdescribed in conjunction with specific embodiments illustrated in theprevious figures. In particular, it may comprise a solvent mixing deviceas hereinbefore described. Typical flow rates of mobile phase as itexits the mobile phase preparation device 520 on the analytical line areon the order of milliliters per minute.

Downstream from the outlet of the mobile phase preparation device 520 aninjector 522 is located, through which a liquid sample is injected intothe continuously flowing mobile phase stream. Once injected, the mobilephase carries the sample into the LC column 524. The column 524 containsthe chromatographic packing material needed to effect the separation asis generally known in the art.

Furthermore, a detector 526 is coupled to the outlet side of the column524 in order to detect the separated compound bands as they elutetherefrom. Possible implementations of detectors 526 include, forinstance, UV absorbance detectors, fluorescence detectors andevaporative light scattering detectors. It is also possible to input theeluate of the LC into a mass spectrometer to analyze the compoundsseparated by liquid chromatography, for example via a spray ionizationsource which transfers analytes of interest from the liquid phase intothe gas phase required for mass spectrometry. In some cases, it mayfurther be useful to couple multiple detectors in series or in parallel.For example, a UV absorbance detector can be used in combination with amass spectrometer. In so doing, an injected sample can be investigatedin two different ways and thus more thoroughly than with only one of theafore-mentioned detection methods alone.

After the detection process, the residual mobile phase exits thedetector 526 and can be sent to waste 528 or collected, for example tobe cleaned and reconditioned, or collected as separated samples forfurther analysis. In principle, all components in the solvent deliverylines, analytical line and dead-ended line, used for pressuremeasurement and purging, have to be equipped and connected with highpressure resistant conduits and high pressure resistant fittings toreliably withstand the high pressure load exerted thereon duringoperation.

Normally, the detector 526 communicates with a computer 530 that recordsthe signal output therefrom and converts it into chromatograms, which inturn can be displayed on a screen or kept accessible for furtherexamination, such as for identifying and quantifying the concentrationof the sample constituents.

A method of operating a mobile phase preparation device during a run ofhigh pressure liquid chromatography is shown in FIG. 6. The methodstarts in step 600 and proceeds to step 602 where a solvent is pumpedfrom a solvent source to an inlet of a manifold. Next, in step 604 thesolvent is passed through a confluence region within the manifold wherethe solvent can be combined with at least one other solvent, beingdelivered to the manifold through a different line and inlet, to form amobile phase. Then, in step 606, the resultant mobile phase is outputfrom the manifold onto an analytical line. In step 608, a portion fromthe flow of mobile phase is branched off at, or downstream from, theconfluence region in order to perform at least one of a pressuremeasurement and venting to exhaust. The method then finishes in step610.

The above description has been focused on applications under ultra-highpressure conditions, that is, at about 6,000 psi (˜4.1×10⁷ Pascal) andmore. However, it goes without saying that the beneficial effectsbrought about by the invention, such as reduced pump compression volumeand, as the case may be, reduced number of high pressure resistantfluidic connections, may also prove valuable in lower pressure regimeswhich are generally considered part of high pressure liquidchromatography. Furthermore, future applications may exceed theultra-high pressure limits typically observed in today's ultra-highpressure liquid chromatography. The present disclosure is therefore notto be construed restrictive in this regard.

It will be understood that various aspects or details of the inventionmay be changed, or various aspects or details of different embodimentsmay be arbitrarily combined, if practicable, without departing from thescope of the invention. Generally, the foregoing description is for thepurpose of illustration only, and not for the purpose of limiting theinvention which is defined solely by the appended claims.

What is claimed is:
 1. A mobile phase preparation device for use with ahigh pressure liquid chromatography system, comprising: a manifoldhaving a plurality of inlets, each inlet fluidly coupled with a solventsource, and the inlets being fluidly coupled to a mobile phase outletthat outputs a mobile phase, composed of at least one solvent, onto ananalytical line; at least one pump for delivering solvent to themanifold; and a manifold branch that is located at a point downstream ofthe plurality of inlets and is fluidly coupled with at least one of apressure sensor and a purge valve.
 2. The device of claim 1, furthercomprising a distribution homogenizer that is fluidly coupled to theanalytical line at the mobile phase outlet and homogenizes aninhomogeneous distribution of solvents in the mobile phase.
 3. Thedevice of claim 2, wherein the distribution homogenizer comprises amixer cartridge.
 4. The device of claim 1, wherein the at least one pumpis a single-piston pump.
 5. The device of claim 1, wherein the pressuresensor is configured to operate up to about 20,000 psi (˜1.4×10⁸Pascal).
 6. The device of claim 1, wherein the purge valve is fluidlycoupled to the pressure sensor.
 7. The device of claim 1, wherein thepressure sensor is a pressure transducer.
 8. The device of claim 7,wherein the pressure transducer comprises a diaphragm sensor thatmeasures fluid displacement.
 9. The device of claim 1, wherein thebranch includes a first outlet connected to the mobile phase output anda second outlet.
 10. The device of claim 9, wherein the manifold has twoinlets for receiving two solvents, the two inlets and the first andsecond outlets being coupled to each other in a cross connection. 11.The device of claim 1, further comprising a controller that operates theat least one pump such that pumping powers for different solvents changeover time as to vary a composition of the mobile phase.
 12. The deviceof claim 1, further comprising tubing that fluidly connects an outlet ofthe at least one pump directly to an inlet of the manifold, whereindimensions of the tubing are minimized as to reduce fluid volume betweenpump and manifold.
 13. The device of claim 1, further comprising acontroller that acquires pressure measurement data from the pressuresensor and adjusts operation of the at least one pump as a function ofthe pressure measurement data.
 14. The device of claim 1, wherein themanifold is constructed as a machined block of material in order tominimize high pressure resistant fluidic connections on fluid lines.15-20. (canceled)